Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: Bone morphogenetic protein (BMP)-4, transforming growth factor (TGF)-β1, serotonin (or 5-hydroxytryptamine; 5-HT), endothelin (ET)-1, and glycogen synthase kinase (GSK)-3β inhibitors increase pulmonary smooth muscle cell size and protein synthesis. A: change in forward scatter in human pulmonary artery smooth muscle cells treated with PBS, BMP-4, TGF-β1, 5-HT, ET-1, LiCl, SB-216763, and EGF. B: overall protein synthesis of cells treated with PBS, BMP-4, TGF-β1, 5-HT, ET-1, LiCl, or SB-216763, as assessed by [3H]leucine incorporation (cpm/well). C: Overall DNA synthesis of cells treated with PBS, BMP-4, TGF-β1, 5-HT, ET-1, LiCl, or SB-216763, as assessed by [3H]thymidine incorporation (cpm/well); n = 3, means ± SE; *P < 0.05, ANOVA.

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: DNA Synthesis

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: Phosphorylation of GSK-3β is required for BMP-4-, TGF-β1-, 5-HT-, and ET-1-induced hypertrophy. A: representative immunoblots for phospho-GSK-3β and total GSK-3β in human pulmonary artery smooth muscle cells treated with BMP-4, TGF-β1, 5-HT, ET-1, LiCl, and SB-216763. B: GSK-3β-A9 was expressed in A7R5 cells via retroviral gene transfer. Expression of GSK-3β-A9 acts as a “dominant-negative,” decreasing the binding of upstream kinases and scaffolding proteins to native GSK-3β. This leads to a relative reduction of phosphorylated, inactive GSK-3β, and an increase in GSK-3β activity. C: effect of GSK-3β-A9 overexpression on the size of cells treated with BMP-4, TGF-β1, 5-HT, ET-1, LiCl, or SB-216763 (*different from MSCV-transduced cells, P < 0.05, ANOVA).

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: Western Blot, Expressing, Dominant Negative Mutation, Binding Assay, Scaffolding, Activity Assay, Over Expression

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: Mechanism of GSK-3β-mediated cell hypertrophy. A: representative immunoblots for phospho- and total eIF2B in pulmonary artery smooth muscle cells treated with BMP-4, TGF-β1, 5-HT, ET-1, and GSK-3β inhibitors. B: effect of BMP-4, TGF-β1, 5-HT, ET-1, LiCl, and SB-216763 on serum response factor (SRF) reporter activity. A7R5 cells were transiently transfected with SV40 Renilla luciferase vector and SRF-luc. Forty-eight hours after treatment, cells were lysed and luciferase activity determined. Each stimulus increased SRF activity (n = 8, means ± SE; *different from control cells, P < 0.05, ANOVA). C: effect of BMP-4, TGF-β1, 5-HT, ET-1, LiCl, and SB-216763 on α-actin mRNA in human pulmonary artery cells. Cells were treated for 4 days and processed for qPCR analysis of α-actin mRNA levels relative to GAPDH mRNA. Each stimulus increased α-actin mRNA (n = 3, means ± SE, *different from control cells, P < 0.05, ANOVA).

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: Western Blot, Activity Assay, Transfection, Luciferase, Plasmid Preparation

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: BMP-4, TGF-β1, 5-HT, and ET-1 activate the p70S6K signaling pathway. A: representative immunoblots for phospho-p70S6K, total p70S6K (top), phospho-S6, and total S6 (bottom) in pulmonary artery smooth muscle cells treated with BMP-4, TGF-β1, 5-HT, and ET-1. B: group mean data (n = 3, ± SE, *different from unstimulated cells, P < 0.05, ANOVA). C: specific siRNAs against p70S6K (top) and S6 (bottom) block phosphorylation of these proteins. D: group mean data (n = 3, ± SE, *different from nontargeting siRNA, P < 0.05, ANOVA).

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: Western Blot, Blocking Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: Activation of the p70S6K pathway is required for cell hypertrophy. Pulmonary artery smooth muscle cells were transfected with either nontargeting siRNA, specific siRNA against p70S6K (A), or siRNA against S6 (B), and treated with BMP-4, TGF-β1, 5-HT, or ET-1. Cell size was measured by flow cytometry. C: representative immunoblots for α-actin and β-actin from cells transfected with either nontargeting siRNA, p70S6K siRNA, or S6 siRNA. D: group mean data for p70S6K siRNA experiments (n = 3, ± SE, *different from nontargeting siRNA, P < 0.05, ANOVA). E: group mean data for S6 siRNA experiments (n = 3, ± SE, *different from nontargeting siRNA, P < 0.05, ANOVA).

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: Activation Assay, Transfection, Flow Cytometry, Western Blot

Journal:

Article Title: Enhanced expression of transient receptor potential channels in idiopathic pulmonary arterial hypertension

doi: 10.1073/pnas.0405908101

Figure Lengend Snippet: The mRNA expression of TRPC channels in NPH lung tissue and PASMC. (A) Representative and summarized (n = 4) mRNA products for TRPC genes and GAPDH in NPH human lung. (B) Representative and summarized (n = 3) data showing TRPC gene mRNA products in human brain tissue (B), PASMC (S), and PAEC (E). M, 100-bp DNA ladder.

Article Snippet: Human PASMC from normal subjects (NPH) were purchased from Cambrex, cultured at 37°C in SMGM, and used at the fourth to sixth passage.

Techniques: Expressing

Journal:

Article Title: Enhanced expression of transient receptor potential channels in idiopathic pulmonary arterial hypertension

doi: 10.1073/pnas.0405908101

Figure Lengend Snippet: TRPC6 mRNA and protein expression is increased in pulmonary tissues from IPAH patients. (A) Representative and summarized (NPH, n = 3; SPH, n = 8; IPAH, n = 3; normalized to α-actin) TRPC6 protein expression in SPH, NPH, and IPAH lung tissues and their α-actin controls. (B) Representative and summarized (n = 3 for each) data showing TRPC6 mRNA products in isolated NPH-, SPH-, or IPAH-PASMC. M, 100-bp DNA ladder. (C) Representative and summarized (n = 5 for each) TRPC6 protein expression in isolated PASMC from NPH, SPH, and IPAH patients. (D) Representative (Top, ×40; Middle, ×100) and summarized immunofluorescence staining of FITC-conjugated TRPC6 (Bottom) in isolated NPH-(n = 11), SPH (n = 21), and IPAH-(n = 36) PASMC. (Scale bars, 20 μm.) ***, P < 0.001 vs. NPH and/or SPH.

Article Snippet: Human PASMC from normal subjects (NPH) were purchased from Cambrex, cultured at 37°C in SMGM, and used at the fourth to sixth passage.

Techniques: Expressing, Isolation, Immunofluorescence, Staining

Journal:

Article Title: Enhanced expression of transient receptor potential channels in idiopathic pulmonary arterial hypertension

doi: 10.1073/pnas.0405908101

Figure Lengend Snippet: Up-regulated TRPC3 expression in IPAH-PASMC. (A) TRPC3 mRNA expression in PASMC from two NPH patients and two IPAH patients. TRPC3 and -4 mRNA expression in PASMC from two IPAH patients and two SPH patients. M, 100-bp DNA ladder. (B) TRPC3 protein expression in the same IPAH and SPH samples as shown in A. (C) Summarized bar graphs (n = 3 for each) depicting normalized TRPC3 mRNA and protein expression. ***, P < 0.001 vs. IPAH.

Article Snippet: Human PASMC from normal subjects (NPH) were purchased from Cambrex, cultured at 37°C in SMGM, and used at the fourth to sixth passage.

Techniques: Expressing

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) Representative immunocytochemistry images of pSPHK2 (pink), actin (green, cytoplasmic marker) and DAPI (blue, nuclear) coimmunostaining in EMAP II treated (2 hr) or vehicle treated fixed hPASMCs, scale bar is 20 μm, n=3. (B) Representative immunoblot probed for pSPHK2, tubulin and lamin B in cytoplasmic and nuclear fractions of hPASMCs following EMAP II treatment for 0, 2 and 4 hours, n=3. (C) Representative immunoblot probed for pSPHK2 and lamin B in nuclear fractions of hPASMCs following EMAP II treatment (150 minutes) with or without SPHK2 inhibitor (D) quantification of nuclear pSPHK2/lamin B, n=3. (E) ELISA-nuclear C18-S1P levels normalized against 1 μg of nuclear proteins in the nuclear fractions of hPASMCs following EMAP II for 15 or 150 minutes with or without SPHK2 inhibitor, n=3 or 4/group. P values are calculated using Kruskal-Wallis against control or Kolmogorov-Smirnov non-parametric test and results are shown as median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Immunocytochemistry, Marker, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) Schematic diagram representing the collection of vascular endothelial cells (ECs) conditioned media (ECM) from ECs grown in 1%O2 or room air to treat vascular smooth muscle cells (SMCs) and, (B) representative dot blot probed for secreted EMAP II expression in ECM. (C) Representative immunoblot probed for KLF4, SPHK2, tubulin, Ac-H3K9 and total histone H3 in whole cell lysates of normoxia or hypoxia ECM with or without EMAP II neutralizing antibody treated hPASMCs pre-transfected with siRNA mediated SPHK2 or scramble silencing and, (D) quantification of KLF4/Tubulin, n=3 and (E) quantification of Ac-H3K9/total histone H3, n=3. (F) KLF4 expression levels normalized against 18S rRNA in normoxia or hypoxia ECM with or without EMAP II neutralizing antibody treated hPASMCs pre-transfected with siRNA mediated SPHK2 or scramble silencing, n=3–4. (G) EMAP II secreted by vascular ECs promote SPHK2/Ac-H3K9/KLF4 signaling in vascular SMCs that may promote PASMCs proliferation. P values are calculated using Kruskal-Wallis against Hy ECM+Scr or Kolmogorov-Smirnov non-parametric test if not mentioned otherwise, and results are shown as median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Dot Blot, Expressing, Western Blot, Transfection

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) Representative immunoblot probed for AIMP1 (precursor form of EMAP II) and Tubulin in protein lysates of human iPAH or FDL, n=19–20/group and, (B) quantitation of AIMP1 (AIMP1/Tubulin) in protein lysates of human iPAH or FDL, n=19–20/group. (C) Representative immunoblot probed for Ac-H3K9, total H3 or tubulin in hPASMCs following EMAP II treatment for 0, 1, 2, 4 and 6 hours and (D) quantitation of Ac-H3K9 expression levels normalized against total H3 in hPASMCs, n=4. (E) Representative immunoblot probed for Ac-H3K9, total H3 or tubulin in hPMVECs following EMAP II treatment for 0, 1, 2, 4 and 6 hours and (F) quantitation of Ac-H3K9 expression levels normalized against total H3 in hPMVECs, n=3. P values are calculated using unpaired t-test or Kolmogorov-Smirnov non-parametric testing and results are shown as means ± SEM or median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Western Blot, Quantitation Assay, Expressing

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) Representative immunoblot probed for Ac-H3K9, total H3, SPHK2 and tubulin in whole cell lysates of hPASMCs following siRNA mediated SPHK2 silencing and post-transfection EMAP II treatment for 4 hours and (B) quantitation of Ac-H3K9/total H3 and (C) quantification of SPHK2/tubulin, n=4. (D) Volcano plot showed the log2-fold changes and statistical significance of hyperacetylated H3K9 regions calculated after differential binding analysis of EMAP II treated vs control hPASMCs. Pink points indicate significantly hyperacetylated H3K9 regions in EMAP II (right to 0) or in control (left to 0). FDR=0.05, n=2 (E) Genome wide distribution of differentially enriched hyperacetylated H3K9 peaks (log2-fold change > 1, p value < 0.05) n=2. (F) Number of peaks of Ac-H3K9 normalized to IgG in with or without SPHK2 inhibitor and EMAP II treated (2–3 hours) hPASMCs, n=2. (G) Gene Ontology results using differentially enriched Ac-H3K9 peaks in EMAP II treated hPASMCs, n=2. (H) Cell proliferation rate in hPASMCs treated with vehicle or EMAP II following SPHK2 inhibitor treatment for 24 hours, n=3. P values are calculated using Kruskal-Wallis against control or Kolmogorov-Smirnov non-parametric test and results are shown as means ± SEM or median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Western Blot, Transfection, Quantitation Assay, Binding Assay, Control, Genome Wide

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) The Venn’s diagram of differential acetylated sites in control vs EMAP II (total) (purple), EMAP II vs iSPHK2+EMAP II (yellow) and control vs EMAPII only in 5’UTR and upstream with fold enrichment greater than 2 (green). The red circle indicates the potential gene set with potential upstream candidate regulatory elements that would be differentially acetylated by EMAP II through SPHK2 in hPASMCs. Venn diagram is created using Venny 2.1 (an online interactive tool), n=2/group (B) Snapshot of IGV view of KLF4 gene in Ac-H3K9 CUT&RUN data of with or without SPHK2 inhibitor and EMAP II treated (2–3 hours) hPASMCs. (cCRE= candidate Cis-Regulatory Elements) n=2/group (C) Representative immunoblot probed for KLF4, SPHK2 and tubulin in whole cell lysates of hPASMCs following siRNA mediated SPHK2 silencing and post-transfection EMAP II treatment for 6–8 hours, and (D) quantitation of KLF4/tubulin and (E) KLF4 expression levels normalized against 18S rRNA in hPASMC cells following siRNA mediated SPHK2 silencing and EMAP II treatment for 6 hours, n=4. P values are calculated using Kolmogorov-Smirnov non-parametric testing and results are shown as median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Control, Western Blot, Transfection, Quantitation Assay, Expressing

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) RNA-seq data of SPHK2, KLF4 and AIMP1 in iPAH: PASMCs and non-iPAH:PASMCs in log2-fold of count per million (cpm). Following two-way ANOVA, Sidak’s multiple comparisons test for logarithmic values, n=4. (B) Representative immunoblot probed for KLF4, SPHK2, tubulin, Ac-H3K9 and total histone H3 in whole cell lysates of non: iPAH or iPAH PASMCs with scramble or SPHK2 siRNA transfection and, quantification of (C) Ac-H3K9/total histone H3 and, (D) KLF4/Tubulin, n=3 (E) KLF4 expression levels normalized against 18S rRNA in non: iPAH or iPAH PASMCs with scramble or SPHK2 siRNA transfection, n=4. (F) Cell proliferation rate of non: iPAH or iPAH PASMC with or without iSPHK2 pretreatment for 24 hours, n=4. (G) The proposed model: Endothelial monocyte activating polypeptide II (EMAP II) plays a key role in reawakening pluripotency factor, KLF4 in human pulmonary artery smooth muscle cells (PASMCs) through stimulation of the nuclear SPHK2/S1P epigenetic modulating axis, suggesting that cooperation between SPHK2 and EMAP II could be a major driving force for epigenetic-mediated vascular PASMCs reprogramming and remodeling in PH. Ablation of SPHK2 expression confers protection against PH by rescuing the global and local transcription machinery from histone acetylation and activation of the pluripotency factor, KLF4. P values are calculated using Kruskal-Wallis against iPAH or Kolmogorov-Smirnov non-parametric test if not mentioned otherwise, and results are shown as median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: RNA Sequencing, Western Blot, Transfection, Expressing, Activation Assay

Journal: Cellular signalling

Article Title: Ligand-mediated dephosphorylation signaling for MAP kinase

doi: 10.1016/j.cellsig.2018.09.005

Figure Lengend Snippet: (A) Cultured human PASMCs or (B) human PAECs were treated with NT or NN (400 nM) for 30 min. Cell lysates were prepared and phosphorylated ERK (pERK) levels were monitored by Western blotting using the pERK1/2 (T202/Y204) antibody. Bar graphs represent means ± SEM of pERK levels determined by densitometry expressed in arbitrary unit (au). *Significantly different from the control value. (N=4 for PASMCs; N=3 for PAECs).

Article Snippet: Cell culture Human PASMCs and human pulmonary artery endothelial cells (PAECs) were purchased from ScienCell Research Laboratories (Carlsbad, CA) and Cell Applications, Inc. (San Diego, CA) and were cultured in accordance with the manufacturers’ instructions in 5% CO 2 at 37°C.

Techniques: Cell Culture, Western Blot, Control

Journal: Cellular signalling

Article Title: Ligand-mediated dephosphorylation signaling for MAP kinase

doi: 10.1016/j.cellsig.2018.09.005

Figure Lengend Snippet: (A) Human PASMCs were treated with neuromedin N (400 nM) for various durations, cell lysates were prepared and phosphorylated forms of ERK (pERK), Stat3 (pStat3), Raf-1 (pRaf-1) and MEK (pMEK) were monitored by Western blotting using pERK1/2 (T202/Y204), pStat3 (Y705), pRaf-1 (S338) and pMEK1/2 (S217/221) antibodies. (B) Bar graphs represent means ± SEM of phospho-protein levels determined by densitometry expressed in arbitrary unit (au). * denotes significant difference from 0 min control at p < 0.01. N=6 (for pERK and pMEK); N=3 (for pRaf-1); N=5 (for pStat3). (C) Total threonine phosphorylation was monitored by Western blotting using the phosphorylated threonine antibody in the cell lysates of human PASMCs treated with neuromedin N (400 nM). (D) HeLa cells were treated with neuromedin N (400 nM) for various durations, cell lysates were prepared and pERK and pMEK were monitored by Western blotting.

Article Snippet: Cell culture Human PASMCs and human pulmonary artery endothelial cells (PAECs) were purchased from ScienCell Research Laboratories (Carlsbad, CA) and Cell Applications, Inc. (San Diego, CA) and were cultured in accordance with the manufacturers’ instructions in 5% CO 2 at 37°C.

Techniques: Western Blot, Control, Phospho-proteomics

Journal: Cellular signalling

Article Title: Ligand-mediated dephosphorylation signaling for MAP kinase

doi: 10.1016/j.cellsig.2018.09.005

Figure Lengend Snippet: Human PASMCs were pre-treated with calyculin A (protein phosphatase type 1 and 2A inhibitor; 50 ng/ml) or juglone (peptidyl-prolyl cis/trans isomerase inhibitor; 20 μM) for 30 min, then treated with neuromedin N for 30 or 60 min. Phosphorylated MEK (pMEK) levels were monitored by Western blotting using the pMEK1/2 (S217/221) antibody. The bar graph represents means ± SEM of pMEK levels determined by densitometry expressed in arbitrary unit (au). * denotes significant difference from control at p < 0.01 (N=3).

Article Snippet: Cell culture Human PASMCs and human pulmonary artery endothelial cells (PAECs) were purchased from ScienCell Research Laboratories (Carlsbad, CA) and Cell Applications, Inc. (San Diego, CA) and were cultured in accordance with the manufacturers’ instructions in 5% CO 2 at 37°C.

Techniques: Western Blot, Control

Journal: Cellular signalling

Article Title: Ligand-mediated dephosphorylation signaling for MAP kinase

doi: 10.1016/j.cellsig.2018.09.005

Figure Lengend Snippet: (A) Expression of NTR types 1, 2 and 3 (NTR1, 2 and 3) were monitored in cell lysates from untreated human PAECs and PASMCs by Western blotting. (B) Lung tissue sections from healthy control rats and rats with pulmonary arterial hypertension (PAH) were subjected to immunohistochemistry using antibodies for NTR1 and NTR2. Representative results of experiments with 5 rats per group are shown. (C) Human PASMCs were treated with control scrambled siRNA or siRNA to knockdown both NTR1 and NTR2. Three days later, cells were treated with neuromedin N (NN; 400 nM) for 30 min. Cell lysates were subjected to Western blotting using the pMEK1/2 (S217/221) antibody to monitor phosphorylated MEK (pMEK). The bar graph represents means ± SEM of pMEK levels determined by densitometry expressed in arbitrary unit (au). * denotes significant difference from untreated control at p < 0.05 (N=3).

Article Snippet: Cell culture Human PASMCs and human pulmonary artery endothelial cells (PAECs) were purchased from ScienCell Research Laboratories (Carlsbad, CA) and Cell Applications, Inc. (San Diego, CA) and were cultured in accordance with the manufacturers’ instructions in 5% CO 2 at 37°C.

Techniques: Expressing, Western Blot, Control, Immunohistochemistry, Knockdown

Journal: Cellular signalling

Article Title: Ligand-mediated dephosphorylation signaling for MAP kinase

doi: 10.1016/j.cellsig.2018.09.005

Figure Lengend Snippet: Human PASMCs were treated with neuromedin N (400 nM). Fix cells were subjected to transmission electron microscopy examinations. (A) Time course of 0, 10 and 30 min. Arrows indicate endocytic vesicles. (B) Transmission electron microscopy images showing neuromedin N-triggered endocytic vesicles (arrows) traveled to the endoplasmic reticulum (ER) cisternae at 30 min. Magnification x 26,000. (C) The transmission electron microscopy images of a larger field to indicate the locations of endocytic vesicles, the enlargement of ER-cisternae along with an increasing number of ribosomes of untreated cell and in the cell treated with neuromedin N for 30 min. N, nucleus. ER, endoplasmic reticulum. R, ribosomes. Magnification x 13,000.

Article Snippet: Cell culture Human PASMCs and human pulmonary artery endothelial cells (PAECs) were purchased from ScienCell Research Laboratories (Carlsbad, CA) and Cell Applications, Inc. (San Diego, CA) and were cultured in accordance with the manufacturers’ instructions in 5% CO 2 at 37°C.

Techniques: Transmission Assay, Electron Microscopy

Journal: Cellular signalling

Article Title: Ligand-mediated dephosphorylation signaling for MAP kinase

doi: 10.1016/j.cellsig.2018.09.005

Figure Lengend Snippet: (A) Human PASMCs were treated with control scrambled siRNA or SNX9 siRNA. Three days later, cells were treated with neuromedin N (NN; 400 nM) for 30 min. Cell lysates were subjected to Western blotting using the pMEK1/2 (S217/221) antibody to monitor phosphorylated MEK (pMEK) as well as to assess the extent of SNX9 knockdown using the SNX9 antibody. The bar graph represents means ± SEM of pMEK levels determined by densitometry expressed in arbitrary unit (au). * denotes significant difference from untreated control at p < 0.05 (N=3). (B) Human PASMCs were treated with control scrambled siRNA, SNX1 siRNA or SNX5 siRNA. Three days later, cells were treated with NN. Cell lysates were subjected to Western blotting to monitor pMEK. (C) Human PASMCs were treated with SNX9 siRNA. Three days later, cells were treated with NN. Fix cells were subjected to transmission electron microscopy examinations.

Article Snippet: Cell culture Human PASMCs and human pulmonary artery endothelial cells (PAECs) were purchased from ScienCell Research Laboratories (Carlsbad, CA) and Cell Applications, Inc. (San Diego, CA) and were cultured in accordance with the manufacturers’ instructions in 5% CO 2 at 37°C.

Techniques: Control, Western Blot, Knockdown, Transmission Assay, Electron Microscopy

Journal: International Journal of Molecular Medicine

Article Title: Reversal of the Warburg effect with DCA in PDGF-treated human PASMC is potentiated by pyruvate dehydrogenase kinase-1 inhibition mediated through blocking Akt/GSK-3β signalling

doi: 10.3892/ijmm.2018.3745

Figure Lengend Snippet: Effect of DCA, LY294002 or combination of DCA and LY294002 on the growth of human PASMCs. The human PASMCs were seeded in 96-well plates in RPMI-1640 medium supplemented with 10% FBS followed by 48 h serum starving prior to exposure to 20 ng/ml PDGF or with increased concentrations of (A) DCA or (B) LY294002 in fresh culture medium with 0.5% foetal bovine serum for 72 h. * P<0.05, ** P<0.01 and *** P<0.001 vs. PDGF-treated cells; #P<0.05 and ## P<0.01 vs. cells treated with 5 mM DCA or 5 µ M LY294002. (C) Cells were exposed to PDGF or 5 µ M LY294002, 10 or 20 mM DCA and combination of 5 µ M LY294002 and DCA (10 mM) for 24 h. * P<0.05, ** P<0.01 as compared with PDGF cells. The data are presented as the mean ± standard deviation of 6 duplicated wells in three separate experiments. PASMCs, pulmonary arterial smooth muscle cells; DCA, dichloroacetate; PDGF, platelet-derived growth factor; D5, D10, D20 and D50, cells treated with dichloroacetate at 5, 10, 20, 50 mM, respectively, following PDGF exposure. L5, 10 and 20, cells treated with LY294002 at 5, 10, 20 µ M, respectively, following PDGF exposure. L5D10, cells treated in a combination of 5 µ M LY294002 and 10 mM dichloroacetate following PDGF exposure.

Article Snippet: Human PASMCs were purchased from Wuxi BioHermes Biomedical Technology Co., Ltd (Wuxi, China).

Techniques: Standard Deviation, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: Reversal of the Warburg effect with DCA in PDGF-treated human PASMC is potentiated by pyruvate dehydrogenase kinase-1 inhibition mediated through blocking Akt/GSK-3β signalling

doi: 10.3892/ijmm.2018.3745

Figure Lengend Snippet: Effect of DCA, LY294002 or combination of DCA and LY294002 on the apoptosis and mitochondria membrane potential of human PASMCs. The PASMCs were seeded into 25 cm 2 tissue culture flask at a density of 5×10 5 cell/flask and cultured in RPMI-1640 complete culture medium for 16 h followed by serum starvation for 24 h. The cells were then exposed to PDGF alone or 5 µ M LY294002, DCA at 10 mM or a combination of 5 µ M LY294002 and 10 mM DCA for 48 h prior to (A) apoptosis or (B) JC-1 assay. (C) The expression levels of caspase-3 and cleaved caspase-3 were analyzed with western blot analysis. The representative change of one of the three experiments is presented, as all assays exhibited identical results. (D) Results were pooled from three separate experiments and are presented as mean ± standard deviation. * P<0.05, ** P<0.01 and *** P<0.001 vs. control cells. ## P<0.01 and ### P<0.001 vs. cells treated with PDGF. C, control; PASMCs, pulmonary arterial smooth muscle cells; DCA, dichloroacetate; PDGF, platelet-derived growth factor; D5 and D10, cells treated with DCA at 5 and 10 mM, respectively, following PDGF exposure; L5, cells treated with LY294002 at 5 µ M following PDGF exposure; L5D10, cells treated with a combination of 5 µ M LY294002 and 10 mM DCA following PDGF exposure.

Article Snippet: Human PASMCs were purchased from Wuxi BioHermes Biomedical Technology Co., Ltd (Wuxi, China).

Techniques: Membrane, Cell Culture, Expressing, Western Blot, Standard Deviation, Control, Derivative Assay

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Rapamycin inhibits mTORC1 and mTORC2. (A) hPASMCs were treated with 100 nM rapamycin for the indicated times and analyzed by immunoblotting for the proteins level of p-p70S6k, p70S6k, p-AKT (S473), p-AKT (T308), AKT. (B) immunoblotting analyses of p-p70S6k, p70S6k, p-AKT (S473), and AKT in hPASMCs, which were stimulated with 5 μg/ml insulin for 24h before treatment with 100 nM rapamycin. (C) hPASMCs were treated with 100 nM rapamycin for the indicated times, and then cell lysates were prepared for and immunoprecipitation (IP) with mTOR antibody. The elution from IP was analyzed by immunoblotting for the levels of mTOR and Rictor. Data are presented as the mean ± SE. One-way ANOVA was used for statistical analysis. NS means not significant. *** p < 0.001; ** p < 0.01; * p < 0.05 versus control.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Western Blot, Immunoprecipitation, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Imatinib inhibits phosphorylation of PDGFRα/β induced by rapamycin in hPASMCs. (A) hPASMCs were treated with 100 nM rapamycin for the indicated times and analyzed by immunoblotting for the proteins level of p-PDGFRα/β, PDGFRα, PDGFRβ. (B) Immunoblotting analyses of p-PDGFRα/β, PDGFRα, PDGFRβ, p-AKT (S473), p-AKT (T308), p-S6 and S6 in hPASMCs treated with vehicle (Control), 100 nM rapamycin (Rap), 5 uM imatinib (Ima) and 100 nM rapamycin + 5 uM imatinib (Rap + Ima) for 48 h. Data are presented as the mean ± SE. One-way ANOVA was used for statistical analysis. NS means not significant. *** p < 0.001, ** p < 0.01, * p < 0.05 versus control.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Effects of rapamycin combined with imatinib on the viability, proliferation and migration of hPASMCs. (A) Cell viability was determined by measuring the absorbance at 0, 24, 48 and 72 h after different drug treatments. (B) A scratch was applied to cell monolayers, and migration of the cells towards the wound was recorded by photomicrographs at 0, 4, and 8h ( n = 3); summarized data showing percent wound closure [(0h wound area–4h or 8h wound area)/0h wound area] * 100%. (C) BrdU assay was performed to determine hPASMCs proliferation under normoxia and hypoxia (3% 0 2 ) for 24 and 48 h. (D) BrdU assay was performed to determine hPASMCs proliferation at 24 and 48 h after different drug treatments. Data are presented as the mean ± SE. Two-way ANOVA was used for statistical analysis. *** p < 0.001; ** p < 0.01; * p < 0.05 versus control; ### p < 0.001, ## p < 0.01, # p < 0.05 versus Rap; $$$ p < 0.001, $$ p < 0.01, $ p < 0.05 versus Ima.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Migration, BrdU Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Rapamycin combined with imatinib attenuates PASMC proliferation and remodeling induced by MCT. (A) H&E staining in lung tissue sections. Summarized data showing pulmonary artery media wall thickness. (B) Lung sections were stained α-SMA (red) and PCNA (green). Yellow arrowheads point at PCNA positive PASMCs and white arrowheads show the vessels. For each of the 5 groups, approximately 600 PASMC nuclei and 100 fields were analyzed. Summarized data showing PCNA positive cells and muscularization. Data are presented as the mean ± SE. One-way ANOVA was used for statistical analysis. *** p < 0.001, ** p < 0.01, * p < 0.05 versus control; ### p < 0.001, ## p < 0.01, # p < 0.05 versus MCT with vehicle; $$$ p < 0.001, $$ p < 0.01, $ p < 0.05 versus MCT with rapamycin.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Rapamycin combined with imatinib attenuates PASMC proliferation and remodeling induced by Hypoxia/Sugen. (A) H&E staining of lung tissue sections and summarized data showing pulmonary artery media wall thickness. (B) Lung sections were stained with α-SMA (red) and PCNA (green). Yellow arrowheads point at PCNA positive PASMCs and white arrowheads show the vessels. For each of the 5 groups, approximately 600 PASMC nuclei and 100 fields were analyzed. Summarized data showing PCNA positive cells and muscularization. Data are presented as the mean ± SE. One-way ANOVA was used for statistical analysis. NS means no significant. *** p < 0.001, ** p < 0.01, * p < 0.05 versus control; ### p < 0.001, ## p < 0.01, # p < 0.05 versus Hypoxia/Sugen with vehicle; $$$ p < 0.001, $$ p < 0.01, $ p < 0.05 versus Hypoxia/Sugen with rapamycin.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Effects of rapamycin combined with imatinib on mTOR and PDGFR signaling pathways in pulmonary artery. (A) Pulmonary artery vessels of were isolated for protein extraction, and the expression of mTORC 1, mTORC 2 and PDGFR signaling pathway related proteins were detected by immunoblotting. Data are presented as the mean ± SE. One-way ANOVA followed by Graphpad prism was used for statistical analysis. NS means no significant. *** p < 0.001; ** p < 0.01; * p < 0.05 versus control. ### p < 0.001; ## p < 0.01; # p < 0.05 versus MCT with vehicle. (B) The schematic representation of the findings of this study: rapamycin chronic treatment in hPASMCs induced the highly expression of phosphorylation of PDGFRs. Imatinib inhibits phosphorylation of PDGFRα/β induced by rapamycin. Abbreviations: GF, growth factors; RTK, receptor tyrosine kinase; PDGF, platelet derived growth factor; PDGFR, platelet derived growth factor receptor; PI3K, phosphoatidylinositol 3-kinase; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5-bisphosphate; mTORC1, mTOR complex 1; mTORC2, mTOR complex 2.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Protein-Protein interactions, Isolation, Protein Extraction, Expressing, Western Blot, Control, Phospho-proteomics, Derivative Assay